Method For Enzmatic Cross-Linking Of A Protein, Cross-Linked Protein Thus Obatained And Use Thereof

Abstract

The present invention provides methods for enzymatic cross-linking of a protein comprising performing a reaction for enzymatic cross-linking of said protein in the presence of a phenolic saccharide, wherein said reaction comprises the step of forming protein-protein cross-links. The present invention further provides a cross-linked protein obtainable by a method according to the invention as well as a protein that is cross-linked by a phenolic saccharide and the use of such proteins in a foodstuff. The invention also provides a foodstuff comprising a cross-linked protein of the invention as well as the use of a phenolic saccharide as a cross-linking agent for proteins.

1 . A method for enzymatic cross-linking of a protein comprising performing a reaction for enzymatic cross-linking of said protein in the presence of a phenolic saccharide, wherein said reaction comprises the step of forming protein-protein cross-links. 
     
     
         2 . Method according to  claim 1 , wherein said step of forming protein-protein cross-links is performed in the presence of a phenolic saccharide which enhances the rate of a protein-protein cross-linking reaction. 
     
     
         3 . Method according to  claim 1 , wherein said step of forming protein-protein cross-links further comprises essentially avoiding the formation of cross-links between the protein and the phenolic saccharide. 
     
     
         4 . Method according to  claim 1 , wherein said phenolic saccharide is an oligosaccharide or monosaccharide. 
     
     
         5 . Method according to  claim 1 , wherein said phenolic saccharide comprises a saccharide chain length of 1-20, preferably of 1-9, more preferably of 1-5 sugar residues. 
     
     
         6 . Method according to  claim 1  wherein said phenolic saccharide has a DP less than 10, more preferably less than 9, even more preferably less than 8, still more preferably less than 7, and yet still more preferably less than 6, 5, 4 or 3. 
     
     
         7 . Method according to  claim 1 , wherein said phenolic saccharide is selected from the group consisting of hydrolyzed arabinoxylan, hydrolyzed pectin, p-coumaroylglucose, feruloylglucose and feruloylarabinose. 
     
     
         8 . Method according to  claim 1 , wherein said phenolic saccharide is selected from the group consisting of hydrolyzed sugar beet pectin and feruloylarabinose. 
     
     
         9 . Method according to  claim 1 , wherein said protein is of animal origin, preferably derived from milk. 
     
     
         10 . Method according to  claim 1 , wherein said protein is a globular protein. 
     
     
         11 . Method according to  claim 1 , wherein said protein is a globular protein selected from the group consisting of β-lactoglobulin, α-lactalbumin and bovine serum albumin. 
     
     
         12 . Method according to  claim 1 , wherein the enzyme for performing said enzymatic cross-linking reaction is laccase, a catechol oxidase, a peroxidase or a tyrosinase. 
     
     
         13 . Method according to  claim 12  wherein the enzyme is laccase. 
     
     
         14 . Method according to  claim 1  wherein the phenolic saccharide is added to the reaction mixture in one step. 
     
     
         15 . Method according to  claim 14  wherein the phenolic saccharide is hydrolysed sugar beet pectin. 
     
     
         16 . Method according to  claim 1  wherein the cross-linking is performed in situ. 
     
     
         17 . Method according to  claim 1  comprising the in situ enzymatic formation of phenolic saccharide. 
     
     
         18 . A cross-linked protein obtainable by a method according to  claim 1 . 
     
     
         19 . A cross-linked protein comprising essentially only protein-protein (homo) cross-links wherein said protein is cross-linked by the action of a phenolic saccharide. 
     
     
         20 . Cross-linked protein according to  claim 19 , wherein said phenolic saccharide is an oligosaccharide or monosaccharide. 
     
     
         21 . Cross-linked protein according to  claim 19 , wherein said phenolic saccharide comprises a saccharide chain length of 1-20, preferably of 1-9, more preferably of 1-5 sugar residues. 
     
     
         22 . Cross-linked protein according to  claim 19 , wherein said phenolic saccharide is selected from the group consisting of hydrolyzed arabinoxylans, hydrolyzed pectins, p-coumaroylglucose, feruloylglucose and feruloylarabinose. 
     
     
         23 . Cross-linked protein according to  claim 19 , wherein said protein is of animal origin, preferably derived from milk. 
     
     
         24 . Cross-linked protein according to  claim 19 -, wherein said protein is a globular protein, preferably selected from the group consisting of β-lactoglobulin, α-lactalbumin, bovine serum albumin and ovalbumin. 
     
     
         25 . Use of a phenolic saccharide for enzymatic cross-linking of a protein to enhance the rate of a protein-protein cross-linking reaction. 
     
     
         26 . Use of a phenolic saccharide in a method for enzymatic cross-linking of proteins, wherein said phenolic saccharide supports the formation of protein-protein (homo) cross-links. 
     
     
         27 . Use according to  claim 25 , wherein said phenolic saccharide is an oligosaccharide or monosaccharide. 
     
     
         28 . Use according to  claim 25 , wherein said phenolic saccharide comprises a saccharide chain length of 1-20, preferably of 1-9, more preferably of 1-5 sugar residues. 
     
     
         29 . Use according to  claim 25 , wherein said phenolic saccharide has a DP less than 10, more preferably less than 9, even more preferably less than 8, still more preferably less than 7, and yet still more preferably less than 6, 5, 4 or 3. 
     
     
         30 . Use according to  claim 25 , wherein said phenolic saccharide is selected from the group consisting of hydrolyzed arabinoxylan, hydrolyzed pectin, p-coumaroylglucose, feruloylglucose and feruloylarabinose. 
     
     
         31 . Use according to  claim 30 , wherein said phenolic saccharide is selected from the group consisting of hydrolyzed sugar beet pectin and feruloylarabinose. 
     
     
         32 . Use according to  claim 25 , wherein said protein is of animal origin, preferably derived from milk. 
     
     
         33 . Use according to  claim 25 , wherein said protein is a globular protein. 
     
     
         34 . Use according to  claim 33 , wherein said protein is a globular protein selected from the group consisting of β-lactoglobulin, α-lactalbumin and bovine serum albumin. 
     
     
         35 . Use according to  claim 25 , wherein the enzyme for performing said enzymatic cross-linking reaction is laccase, a catechol oxidase, a peroxidase or a tyrosinase. 
     
     
         36 . Use according to  claim 35 , wherein the enzyme is laccase. 
     
     
         37 . Use of a cross-linked protein according to  claim 18  in a foodstuff, preferably a foodstuff selected from the group consisting of beer foam, puddings, custards, bread, cheeses, ice cream, spreads, toppings, margarine, mayonnaise, sauces, dressings, sausages, hams, other meat products, tofu, noodles, yogurts, frozen desserts, icings, sour creams, bakery creams, batters, batter coatings, baked products, creamers, shortenings, baby foods, powdered soups, liquid soups and breadings. 
     
     
         38 . Product comprising a cross-linked protein according to  claim 18 , preferably a product selected from a coacervate, a gel, a coating or a proteinaceous fibre comprising product. 
     
     
         39 . Method according to  claim 2 , wherein:
 said phenolic saccharide is an oligosaccharide or monosaccharide;   said phenolic saccharide comprises a saccharide chain length of 1-20, preferably of 1-9, more preferably of 1-5 sugar residues;   said phenolic saccharide has a DP less than 10, more preferably less than 9, even more preferably less than 8, still more preferably less than 7, and yet still more preferably less than 6, 5, 4 or 3;   said phenolic saccharide is selected from the group consisting of hydrolyzed arabinoxylan, hydrolyzed pectin, p-coumaroylglucose, feruloylglucose and feruloylarabinose;   said phenolic saccharide is selected from the group consisting of hydrolyzed sugar beet pectin and feruloylarabinose;   said protein is of animal origin, preferably derived from milk;   said protein is a globular protein;   said protein is a globular protein selected from the group consisting of β-lactoglobulin, α-lactalbumin and bovine serum albumin;   the enzyme for performing said enzymatic cross-linking reaction is laccase, a catechol oxidase, a peroxidase or a tyrosinase;   the enzyme is laccase;   the phenolic saccharide is added to the reaction mixture in one step;   the phenolic saccharide is hydrolysed sugar beet pectin;   the cross-linking is performed in situ;   and further comprising the in situ enzymatic formation of phenolic saccharide.   
     
     
         40 . A cross-linked protein obtainable by a method according to  claim 39 . 
     
     
         41 . Cross-linked protein according to  claim 20 , wherein:
 said phenolic saccharide comprises a saccharide chain length of 1-20, preferably of 1-9, more preferably of 1-5 sugar residues;   said phenolic saccharide is selected from the group consisting of hydrolyzed arabinoxylans, hydrolyzed pectins, p-coumaroylglucose, feruloylglucose and feruloylarabinose;   said protein is of animal origin, preferably derived from milk;   said protein is a globular protein, preferably selected from the group consisting of β-lactoglobulin, α-lactalbumin, bovine serum albumin and ovalbumin.   
     
     
         42 . Use according to  claim 26 , wherein:
 said phenolic saccharide is an oligosaccharide or monosaccharide;   said phenolic saccharide comprises a saccharide chain length of 1-20, preferably of 1-9, more preferably of 1-5 sugar residues;   said phenolic saccharide has a DP less than 10, more preferably less than 9, even more preferably less than 8, still more preferably less than 7, and yet still more preferably less than 6, 5, 4 or 3;   said phenolic saccharide is selected from the group consisting of hydrolyzed arabinoxylan, hydrolyzed pectin, p-coumaroylglucose, feruloylglucose and feruloylarabinose;   said phenolic saccharide is selected from the group consisting of hydrolyzed sugar beet pectin and feruloylarabinose;   said protein is of animal origin, preferably derived from milk;   said protein is a globular protein;   said protein is a globular protein selected from the group consisting of β-lactoglobulin, α-lactalbumin and bovine serum albumin;   the enzyme for performing said enzymatic cross-linking reaction is laccase, a catechol oxidase, a peroxidase or a tyrosinase;   the enzyme is laccase.   
     
     
         43 . Use of a cross-linked protein according to  claim 19  in a foodstuff, preferably a foodstuff selected from the group consisting of beer foam, puddings, custards, bread, cheeses, ice cream, spreads, toppings, margarine, mayonnaise, sauces, dressings, sausages, hams, other meat products, tofu, noodles, yogurts, frozen desserts, icings, sour creams, bakery creams, batters, batter coatings, baked products, creamers, shortenings, baby foods, powdered soups, liquid soups and breadings. 
     
     
         44 . Product comprising a cross-linked protein according to  claim 19 , preferably a product selected from a coacervate, a gel, a coating or a proteinaceous fibre comprising product.